US5407803A - Method for measuring the activity of angiotensin converting enzyme in biological samples - Google Patents

Method for measuring the activity of angiotensin converting enzyme in biological samples Download PDF

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US5407803A
US5407803A US07/830,341 US83034192A US5407803A US 5407803 A US5407803 A US 5407803A US 83034192 A US83034192 A US 83034192A US 5407803 A US5407803 A US 5407803A
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ang
ace
sample
angiotensin
activity
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Hans R. Brunner
Juerg Nussberger
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B M R A Corp BV
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/804—Radioisotope, e.g. radioimmunoassay

Definitions

  • the present invention concerns a new method for measuring the enzyme activity of angiotensin converting enzyme (ACE) in biological samples using angiotensin I as a substrate and a monoclonal antibody for trapping angiotensin II formed from angiotensin I; eventually the angiotensin II is detected by radioimmunoassay (RIA).
  • ACE angiotensin converting enzyme
  • activity of the angiotensin converting enzyme is usually measured in vitro by estimation of products cleaved by the enzyme from synthetic substrates.
  • substrates have affinities for ACE different from the natural substrate angiotensin I, and insensitive detection systems necessitate millimolar substrate concentrations while physiological angiotensin I concentrations are in the picomolar range.
  • Plasma ACE-activity is measured for drug monitoring in patients treated with ACE-inhibitors and conventional methods used so far provided well reproducible results.
  • absolute ACE-activities were found to differ considerably (accuracy) when measured in a given plasma with different methods (Juillerat L., Nussberger J., Menard J., Mooser V., Waeber B., Graf P., Brunner H. R., Hypertension 1990; 12: 87-92).
  • the ratio of plasma concentrations of angiotensin (Ang) II to Ang I is used as an indicator of ACE-activity in vivo (Giese J., Rasmussen S., Damkjaer M.
  • Inaccuracy of conventional methods may be due to several reasons: Firstly, substrates used are chemically different from the natural substrate angiotensin I; secondly, insensitive detection systems used to quantitate products cleaved by ACE from these substrates necessitate very high substrate concentrations; thirdly, dilution of biological samples in vitro induces reaction conditions different from those in vivo.
  • the object of the present invention is to provide a new and accurate method of measurement of ACE-activity which overcomes the above mentioned difficulties.
  • the subject of the present invention is thus the method according to the definition of claim 1.
  • plasma ACE-activity is estimated in minimally diluted specimens, using the natural substrate angiotensin I at close to physiological concentration. Femtomoles of generated angiotensin II are trapped by specific high affinity monoclonal antibodies and thus protected from degradation by angiotensinases during the incubation step. The same antibodies are subsequently used for quantitation by radioimmunoassay (Poulsen K, Jorgensen J. J. Clin. Endocrinol. Metab. 1974; 39: 816-825).
  • the new assay for ACE-activity measurement quantitates reliably femtomoles of generated angiotensin II in plasma from angiotensin I added at 10 picomolar concentration.
  • the detection limit was 20 fmol/ml/min.
  • Normal human plasma had an ACE-activity of 335 ⁇ 83 fmol/ml/min (mean ⁇ SD).
  • Precision was characterized by coefficients of variation of less than 11% both within-assay and between-assays.
  • FIG. 1 shows a graph wherein the linearity of generation of angiotensin II in a plasma sample incubated at 37 ° C. is demonstrated: generated angiotensin II is trapped by added high affinity monoclonal antibodies and protected against enzymatic degradation. Subsequent dilution makes it possible to quantitate angiotensin II radioimmunologically by using these antibodies.
  • ACE converting enzyme
  • In vitro measurement by conventional procedures using Z-Phe-His-Leu as a substrate (FIG. 2a) underestimated ACE-inhibition whereas the new trapping method (FIG. 2b) indicated similar ACE-activity in vitro (A) as estimated in vivo (Q) by the ratio of plasma angiotensin II to angiotensin I.
  • FIG. 3 is a graph showing the in vivo (dashed line) and in vitro (solid lines) plasma converting enzyme (ACE) activity in 10 normal men before and up to 24 hours after ingestion of 20 mg enalapril.
  • In vivo ACE-activity was estimated by the ratio of plasma angiotensin II to angiotensin I (Ang II/Ang I).
  • In vitro measurement using the new trapping assay gave virtually identical results as those measured in vivo.
  • ACE-activity For the measurement of ACE-activity, blood samples (5 ml) were collected from the antecubital vein into prechilled glass tubes containing heparin (56 USP) and 25 ⁇ l renin inhibitor CGP 29287 (Ciba-Geigy, Basel, Switzerland) at 1 mM in water. Blood was centrifuged at 4° C. and 2000 g for 10 minutes and plasma aliquots of 0.5 ml were snap frozen in liquid nitrogen and stored at -70° C. until analyzed.
  • Monoclonal antibodies against angiotensin (Ang)II can be produced by the somatic cell fusion technique (Kohler G., Milstein C., Nature 1975; 256: 495-497) as previously described in detail (Nussberger J., Mudgett-Hunter M., Matsueda G. R., Haber E., Hybridoma 1984; 3: 373-376) but using SP 2/0 myeloma cells and spleen lymphocytes of a C3H mouse hyperimmunized with Ang II which was coupled by its carboxy-terminus to bovine thyroglobulin (Nussberger J., Matsueda G. R., Re R., Haber E., J. Immunol.
  • angiotensinase inhibitors such as tripotassium ethylenediaminetetraacetate (EDTA, purchased from Fluka, Buchs, Switzerland) or bacitracin (Grossmann Pharmaca, Basel, Switzerland).
  • Albumin buffer consisted of a 0.1M tris buffer (Sigma, St. Louis, Mo.) containing per liter 5 gram heat inactivated bovine serum albumin (Sigma), 20 mmol bacitracin and 200 mg sodium azide (Merck, Darmstadt, Germany). The pH was adjusted to 7.5 at room temperature.
  • Angiotensin I and angiotensin II standard peptides were purchased from Peninsula Labs (St. Helens, England). 125 I-Ang II was obtained from New England Nuclear Inc. (Boston, Mass.).
  • Ten microliters of monoclonal antibody TRAP (which was secreted from the Hybridoma cell line TRAP DSM ACC2003) was added (mouse ascites diluted 1:8400 in 3M tris buffer at pH 7.3).
  • antibody-bound and free Ang II were separated by adding 0.3 ml water containing 2% dextran-coated charcoal, mixing for 10 minutes, centrifugation at 4° C. (for 20 minutes at 6000 g) and decanting of the supernatant. Supernatant and pellet were counted in a ten-well scintillation counter (GammamasterR, Wallac, Turku, Finland) and generated Ang II was read from a simultaneously established standard curve.
  • Plasma ACE-activity was also measured by an established method using as substrate synthetic carbobenzoxy-Phe-His-Leu at 1.8 mM concentration (Piquilloud Y., Reinharz A., Roth M. Biochem. Biophys. Acta 1970; 206: 136-142). The ratio of the plasma concentrations Ang II/Ang I was used for estimation of ACE-activity "in vivo" (Nussberger J. , Juillerat L., Perret F., Waeber B., Bellet M., Brunner H. R., Menard J., Am Heart J. 1989; 117: 717-722).
  • Sensitivity The present assay is optimized for a final dilution of the monoclonal antibody at 1:924000.
  • the smallest amount of unlabeled Ang II (16 fmol/tube) added to the standard curve results in antibody-binding of 50% of the tracer-angiotensin. This is more than two standard deviations less than tracer binding in the absence of unlabeled Ang II.
  • the smallest possible amount of Ang II which can be read from the standard slope is 16 fmol.
  • the theoretical detection limit for ACE-activity in a 40 ⁇ l plasma sample which was incubated for 20 minutes at 37 ° C. is 20 fmol/ml/min (16 ⁇ 25/20).
  • the actual detection limit is zero, since EDTA-blanks may contain more than 16 fmol Ang II and these blanks are subtracted from Ang II generated during the incubation.
  • ACE is a relatively unspecific enzyme since it cleaves substrates other than Ang I such as bradykinin or substance P.
  • the present assay measures specifically Ang II (carboxy-terminal-specific monoclonal antibody) and uses Ang I as substrate under almost physiological conditions. Endogenous Ang II is subtracted as well as Ang II generated in plasma despite renin inhibition and ACE-inhibition by EDTA (blanks).
  • Plasma ACE-activity in 22 normal human subjects was found to be 334.7 ⁇ 82.9 fmol/ml/min (Mean ⁇ SD) with a range between 178 and 475.
  • Table 1 demonstrates the effect of the single oral dose of 20 mg enalapril in 10 volunteers on plasma ACE-activity, plasma levels of Ang I and Ang II as well as the ratio of plasma Ang II to Ang I. All parameters reached peak effects at 4 and 8 hours after enalapril administration.
  • ACE-activities were expressed for every subject as percent of baseline ACE-activity and means ⁇ SEM were calculated for each time point (FIG. 2).
  • In vivo ACE-activity ratio of plasma Ang II to Ang I
  • minima at 4 and 8 hours post enalapril intake with 2.1 ⁇ 0.3 and 2.0 ⁇ 0.4% and on the following morning (24 hours) in vivo ACE-activity was still only 21.9 ⁇ 5.0%. This was in excellent agreement with ACE-activities measured in vitro with the trapping-assay: 3.6 ⁇ 2.4, 1.3 ⁇ 1.3 and 27.4 ⁇ 3.4%.
  • the ratio of plasma Ang II to Ang I was used to estimate in vivo ACE-activity: It decreased by 98% at peak inhibition and returned to 22% ACE-activity at 24 hours. Very similar results were obtained for plasma ACE-activity measured in vitro with the method according to the invention based on trapping by monoclonal antibodies of generated Ang II (decrease by 99% at peak inhibition and return to 27% ACE-activity at 24 hours). In contrast, the conventional method of ACE-activity estimation provided different results: Peak ACE-inhibition with only 83% decrease from baseline and substantial return towards baseline with 66% ACE-activity at 24 hours. Thus, the new method of ACE-activity measurement according to the invention provided more accurate results than the established method, if the ratio Ang II/Ang I is accepted as a standard.
  • the new method of ACE-activity measurement in vitro compares favorably with established procedures: Femtomoles rather than nanomoles can be detected which may be helpful for comparison of peak effects of different ACE-inhibitors.
  • the specificity of the procedure is also greatly enhanced since it is based on a monoclonal antibody of unique specificity and affinity for Ang II and the natural substrate Ang I is used under nearly physiological conditions (conventional methods use diluted plasma and extremely high concentrations of substrates of affinities different from that of Ang I and in the presence of high-affinity ACE-inhibitors which may lead to underestimation or overestimation of ACE-inhibition).
  • the Ang I concentration in almost undiluted plasma of the trapping assay is sufficient to warrant linearity of the Ang II production during the incubation step as demonstrated in FIG. 2.
  • the precision of the trapping method is in our hands not better than that of the conventional techniques, but coefficients of variation below 11% for both within- and between-assay precision certainly demonstrate the validity of the new procedure.
  • the high accuracy appears to be a major advantage of the trapping method.
  • equipment needed for the new procedure is that of a routine radioimmunoassay laboratory including a gammacounter.
  • Monoclonal antibodies are obtainable according to Nussberger et al., Hybridoma 1984, 3: 373-376, a monoclonal antibody was deposited at DSM under the above indicated deposition number, other antibodies against Ang II, and labeled and unlabeled angiotensins are commercially available.
  • the present invention provides a reliable and accurate new method for the measurement of plasma ACE-activity which is based on trapping of generated Ang II during the enzymatic reaction by high-affinity monoclonal antibodies which are subsequently used to quantitate the peptide by radioimmunoassay.

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US07/830,341 1991-02-01 1992-01-31 Method for measuring the activity of angiotensin converting enzyme in biological samples Expired - Fee Related US5407803A (en)

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EP91810074A EP0499010B1 (de) 1991-02-01 1991-02-01 Verfahren zur Messung der Aktivität angiotensinkonvertierenden Enzyms in biologischen Proben
EP91810074 1991-02-01

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635359A (en) * 1991-02-01 1997-06-03 B.M.R.A. Corporation B.V. Method for measuring the activity of angiotensin converting enzyme in biological samples
US20030100029A1 (en) * 2001-11-09 2003-05-29 Clinical Reference Laboratory Methods of determining active levels of drugs in fluid samples
CN110031633A (zh) * 2019-03-30 2019-07-19 山西农业大学 不同毛色绵羊皮肤中血管紧张素ⅱ的表达试验方法

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DE60017211T2 (de) 1999-02-17 2005-06-02 Pfizer Products Inc., Groton Fusionsproteine mit Trägern welche eine doppelte Immunantwort induzieren

Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0273453A2 (de) * 1987-01-02 1988-07-06 The Du Pont Merck Pharmaceutical Company Hybridoma-Zellinien und monoklonale Antikörper gegen Angiotensin-II

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Publication number Priority date Publication date Assignee Title
EP0499010B1 (de) * 1991-02-01 1996-09-04 B.M.R.A. Corporation B.V. Verfahren zur Messung der Aktivität angiotensinkonvertierenden Enzyms in biologischen Proben

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0273453A2 (de) * 1987-01-02 1988-07-06 The Du Pont Merck Pharmaceutical Company Hybridoma-Zellinien und monoklonale Antikörper gegen Angiotensin-II

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Knud Poulsen, "Simplified Method for Radioimmunoassy of Enzyme Systems" The Journal of Laboratory and Clinical Medicine 78:309-315, 1971.
Knud Poulsen, Simplified Method for Radioimmunoassy of Enzyme Systems The Journal of Laboratory and Clinical Medicine 78:309 315, 1971. *
Nussberger et al., "Angiotensin II Measurement with High-Affinity Monoclonal Antibodies" Journal of Hypertension 6 Suppl 4:S424-S425, 1988.
Nussberger et al., Am. J. Hypertension 5: 393 398, 1992. *
Nussberger et al., Am. J. Hypertension 5: 393-398, 1992.
Nussberger et al., Angiotensin II Measurement with High Affinity Monoclonal Antibodies Journal of Hypertension 6 Suppl 4:S424 S425, 1988. *
Poulsen and Jorgensen, "An Easy Radioimmunological Microassay of Renin Activity, Concentration and Substrate in Human and Animal Plasma and Tissues Based on Angiotensin I Trapping by Antibody" The Journal of Clinical Endocrinology and Metabolism 39:816-825, 1974.
Poulsen and Jorgensen, An Easy Radioimmunological Microassay of Renin Activity, Concentration and Substrate in Human and Animal Plasma and Tissues Based on Angiotensin I Trapping by Antibody The Journal of Clinical Endocrinology and Metabolism 39:816 825, 1974. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635359A (en) * 1991-02-01 1997-06-03 B.M.R.A. Corporation B.V. Method for measuring the activity of angiotensin converting enzyme in biological samples
US20030100029A1 (en) * 2001-11-09 2003-05-29 Clinical Reference Laboratory Methods of determining active levels of drugs in fluid samples
US7094558B2 (en) * 2001-11-09 2006-08-22 Clinical Reference Laboratory Methods of determining active levels of drugs in fluid samples
CN110031633A (zh) * 2019-03-30 2019-07-19 山西农业大学 不同毛色绵羊皮肤中血管紧张素ⅱ的表达试验方法

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ATE142343T1 (de) 1996-09-15
EP0499010A1 (de) 1992-08-19
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US5635359A (en) 1997-06-03
DE69121877D1 (de) 1996-10-10
JPH05192182A (ja) 1993-08-03

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